Nuclear Medicine and Biology 49 (2017) 8–15

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

Relationship between [ 14C]MeAIB uptake and amino acid transporterfamily gene expression levels or proliferative activity in a pilot study inhuman carcinoma cells: Comparison with [ 3H]methionine uptake
Shinya Kagawa a,b, Ryuichi Nishii a,c, Tatsuya Higashi a,c, Hiroshi Yamauchi a, Emi Ogawa d, Hiroyuki Okudaira e,
Masato Kobayashi f, Mitsuyoshi Yoshimoto g, Naoto Shikano h, Keiichi Kawai b,⁎a

Division of PET Imaging, Shiga Medical Center Research Institute, Shiga, Japan
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japanc
Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, Chiba, Japand
Department of Radiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japane
Research Center, Nihon Medi-Physics Co., Ltd., Chiba, Japanf
Wellness Promotion Science Center, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa, Japang
Division of Functional Imaging, Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Chiba, Japanh
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japanba r t i c l e

i n f oArticle history:
Received 26 October 2016
Received in revised form 18 January 2017
Accepted 18 January 2017
Keywords:
MeAIB
Amino acid transporters
System A
Met
Flt
Proliferative activity

a b s t r a c t
Introduction: To clarify the difference between system A and L amino acid transport imaging in PET clinicalimaging, we focused on the use of α-[N-methyl-11C]-methylaminoisobutyric acid ([11C]MeAIB), and compared
it with [S-methyl- 11C]-L-methionine ([ 11C]MET). The aim of this study was to assess the correlation of
accumulation of these two radioactive amino acid analogs with expression of amino acid transporters and cellproliferative activity in carcinoma cells.
Methods: Amino acid uptake inhibitor studies were performed in four human carcinoma cells (epidermal
carcinoma A431, colorectal carcinoma LS180, and lung carcinomas PC14/GL and H441/GL) using the radioisotope
analogs [3H]MET and [14C]MeAIB. MeAIB was used to inhibit the A system and 2-amino-2-norbornane-carboxylic
acid (BCH) was used to inhibit the L system. The carcinoma gene expression levels of a number of amino acidtransporters were measured by microarray and quantitative polymerase chain reaction. Carcinoma proliferative
activity was assessed using accumulation of [methyl-3H]-3'-deoxy-3'-ﬂuorothymidine ([3H]FLT).
Results and conclusion: [14C]MeAIB uptake occurred principally via a Na+-dependent A type mechanism whereas[3H]MET uptake occurred predominantly via a Na+-independent L type mechanism although other transporters
were also utilized depending on cell type. There was no correlation between [3H]MET uptake and total system L
amino acid transporter (LAT) expression. In contrast, [14C]MeAIB uptake strongly correlated with total system A
amino acid transporter (SNAT) expression and proliferative activity in this preliminary study using four humancarcinoma cell lines. Carcinoma proliferative activity also correlated with total SNAT expression.
Advances in Knowledge and Implications for Patient Care: Because there is a signiﬁcant correlation between theaccumulation of [14C]MeAIB and the gene expression level of total SNAT as well as the accumulation of [3H]FLT, it
is suggested that use of the analog [ 11C]MeAIB in PET may provide an indication of tumor cell proliferative
activity. [11C]MeAIB is therefore expected to be very useful in PET imaging.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction
2-[18F]ﬂuoro-2-deoxy-D-glucose ([18F]FDG), an analog of glucose, isthe most commonly used radiopharmaceutical in positron emission tomography (PET)-CT imaging [1]. PET-CT imaging is based on the preferential uptake of [ 18F]FDG in tumor cells as compared to normal cells,⁎ Corresponding author at: Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Odatsuno, Kanazawa city, Ishikawa 920-0942, Japan.
Tel.: +81 76 265 2527; fax: +81 76 234 4366.
E-mail address: kei@mhs.mp.kanazawa-u.ac.jp (K. Kawai).
http://dx.doi.org/10.1016/j.nucmedbio.2017.01.008
0969-8051/© 2017 Elsevier Inc. All rights reserved.

because glucose metabolism is increased in tumor cells. [ 18F]FDG PETCT has been found to be useful in lesion detection and characterization,
evaluation of tumor stage, assessment of treatment response and detection of recurrent disease [2,3]. However, the speciﬁcity of this technique
is low in patients with active infections and inﬂammatory diseases(because of high FDG uptake in macrophages) and in the brain (becauseof high background FDG uptake) [4,5]. Therefore, the development of
post-FDG radiopharmaceuticals is needed.
After glucose transport, amino acid transport is another important
pathway in cellular energy metabolism. Therefore, natural or artiﬁcialamino acid analogs have been widely studied clinically as potential

S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

post-FDG radiopharmaceuticals for PET imaging; one of the mostimportant radiolabeled amino acids in this regard is [S-methyl-11C]-Lmethionine ([ 11C]MET) [6,7]. Since both amino acid transport andprotein synthesis rates are enhanced in tumors, [ 11C]MET has been as
widely used in brain tumor imaging as O-(2-[ 18F]-ﬂuoroethyl)-Ltyrosine ([18F]FET) [8,9].
Numerous amino acid transporters have been identiﬁed at the molecular level and have been characterized in mammalian cells [10–12].
The main transport systems for the uptake of neutral amino acids are
the A, L, and alanine-serine-cysteine (ASC) amino acid transport systems. System L amino acid transporters are Na+ independent, and are
the main transport mechanism for methionine, tyrosine, phenylalanine,
and their analogs such as FAMT (3-ﬂuoro-α-methyl-tyrosine) [13–16].
The system A and ASC amino acid transporters are Na +-dependent,
however, compared to the system L transporters, their involvement in
the transport by radiolabeled amino acids in nuclear medicine has notbeen studied in detail.
The artiﬁcial amino acid radiopharmaceutical α-[N-methyl- 11C]methylaminoisobutyric acid ([ 11C]MeAIB) is a promising speciﬁc substrate of system A amino acid transport. Compared with [ 11C]MET,[ 11C]MeAIB is metabolically stable [17] and it has been studied bothpre-clinically and clinically. For example, [ 11C]MeAIB has been shown
to be useful in the measurement of amino acid uptake into skeletal
muscle and in the diagnosis of malignant lymphoma and head andneck cancers [18–20]. In our institute, [ 11C]MeAIB PET has proven usefulin the diagnosis of chest diseases, especially in the differential diagnosis
between sarcoidosis and metastasis [21].
Previous studies in carcinoma cells have shown that there is a highcorrelation between both MET and FAMT uptake and the gene expression levels of system L amino acid transporters [15,16,22]. However,
the relationship between the accumulation of radiolabeled aminoacids and the gene expression levels of system A amino acid transporters has not been examined.
In this study, we explored the amino acid transport systems in four different human carcinoma cell lines using α-[1-14C]-methylaminoisobutyricacid ([14C]MeAIB) as a substrate and comparing it to [S-methyl-3H]-Lmethionine ([3H]MET) and [methyl-3H]-3'-deoxy-3'-ﬂuorothymidine([3H]FLT). We elected to use 14C or 3H–labeled amino acid analogs
because both [ 11C]MeAIB and [ 11C]MET have a very short half-life(20 min). We also investigated the gene expression proﬁles of numerous
amino acid transporters in these four types of human carcinoma celllines using microarray analysis. Following on from this initial screen weused quantitative reverse transcription polymerase chain reaction (qRTPCR) to characterize the expression of the mRNAs encoding SNAT1,
SNAT2, SNAT4, ASCT1, ASCT2, y+LAT1, y+LAT2, LAT1, LAT2, LAT3, LAT4,
and 4F2hc. We then examined the correlation between accumulation of[3H]MET and the quantitative mRNA expression of total system L transporter (total LAT = LAT1 + LAT2 + LAT3 + LAT4) and total system A
amino acid transporters (total SNAT = SNAT1 + SNAT2 + SNAT4) inthese human carcinoma cell lines. Finally, we examined the correlation
between accumulation of [3H]FLT uptake and [14C]MeAIB uptake as wellas the correlation between [3H]FLT uptake and the quantitative gene expression of total SNAT transporters.
2. Materials and methods
2.1. Radiolabeled amino acid analogs and amino acid transport inhibitors
Because of the short half-life of 11C (20 min), radiolabeled aminoacid analogs with much longer half-lives were used instead. 14C–labeled
MeAIB ([14C]MeAIB, 37 kBq/ml), 3H–labeled MET ([3H]MET, 18.5 kBq/ml) and 3H–labeled FLT ([ 3H]FLT, 18.5 kBq/ml) were obtained from
American Radiolabeled Chemicals Inc. (St Louis, Missouri, USA). MeAIB(α-methylaminoisobutyric acid), a speciﬁc inhibitor of system A, andBCH (2-amino-2-norbornane-carboxylic acid), a speciﬁc inhibitor ofsystem L, were acquired from Sigma-Aldrich Japan KK (Tokyo, Japan).

92.2. Cell culture
Cell line studies were performed using a modiﬁcation of themethods described by Shikano et al. and Nakajima et al. [23,24], as follows. A431 and LS180 cell lines were purchased from Dainippon
Sumitomo Pharma Co., Ltd. (Osaka, Japan). The cultured human tumor
cell lines H441 and PC14 were obtained from the University of Texas,
MD Anderson Cancer Center, Houston, TX, USA. All cells were cultured
in 150 mm cell culture dishes (Becton Dickinson, New Jersey, USA)in a
5% CO2 humidiﬁed atmosphere at 37 °C with speciﬁc media as follows;
A431 cells were maintained in Dulbecco's modiﬁed Eagle”'s medium(DMEM; Sigma-Aldrich, Japan) containing high glucose supplemented
with 10% fetal bovine serum (FBS) and 3.7 g/L NaHCO3, H441 cells
were maintained in RPMI-1640 medium (Sigma-Aldrich, Japan) supplemented with 10% FBS, 1% sodium pyruvate (Sigma-Aldrich,Japan) and 2.0 g/L NaHCO3, PC14 cells were maintained in DMEM/
Nutrient Mixture F-12 Ham (DMEM/F12; Sigma-Aldrich, Japan) supplemented with 10% FBS and 1.2 g/L NaHCO3, and LS180 cells weremaintained in minimum essential medium Eagle (MEM; SigmaAldrich, Japan) supplemented with 10% FBS, 1% sodium pyruvateand 2.2 g/L NaHCO3. Sub-culturing was performed every ﬁve days
using 0.02% EDTA and 0.05% trypsin. For amino acid uptake experiments, cells were seeded into a 24-well Multiwell Plate (Becton
Dickinson, New Jersey, USA) at a density of 5 × 10 5 cells/well andwere used 24 h after plating.

2.3. Measurement of [14C]MeAIB, [3H]MET and [3H]FLT transport inhuman carcinoma cells
For transport studies in a sodium-containing medium, phosphatebuffered saline (PBS) pH 7.4 (137 mM NaCl, 3 mM KCl, 8 mM
Na2HPO4, 1.5 mM K2HPO4, 1 mM CaCl2 and 0.5 mM MgCl2) was used.
For sodium-free transport studies, the NaCl and Na2HPO4 in PBS were
replaced with the same concentrations of choline-Cl and K2HPO4, respectively. After removal of the culture medium, the 24-well Multiwell
Plate was washed once with 5 mL of incubation medium for 10 min at
37 °C. The cells were then incubated with 500 μL/well of incubation medium containing [ 14C]MeAIB (37 kBq/mL) and [ 3H]MET (18.5 kBq/mL)or [ 3H]FLT (18.5 kBq/mL) for 10 min at 37 °C. For the experiment involving amino acid transport, inhibitors were added to a ﬁnal concentration of 1 mM, and the cells were then incubated for 10 min at 37 °C
with [ 14C]MeAIB (37 kBq/mL) and [ 3H]MET (18.5 kBq/mL) or [ 3H]
FLT (18.5 kBq/mL). After incubation with the radiolabeled aminoacid analog, the medium was aspirated and the monolayers wererapidly rinsed twice with 500 μL of ice-cold incubation medium.
Cells were solubilized in 500 μL of 0.1 N NaOH, and the radioactivity(either 3H and 14C) of an aliquot (400 μL) was measured by additionof Clear-Sol II (Nacalai Tesque Inc., Kyoto, Japan) and scintillation
counting using an LSC-5100 liquid scintillation counter (Hitachi
Aloka Medical, Ltd., Tokyo, Japan).
To characterize relative contributions of each type of transportsystem to overall amino acid analog uptake, we performed inhibition
experiments with inhibitors as described above. BCH was used as a system L inhibitor and MeAIB was used as a system A inhibitor. To calculate
the relative contributions of amino acid transporter systems, we used
the methods reported by Shikano et al. [23] and Nakajima et al. [24].
In brief, uptake of [3H]MET and [ 14C]MeAIB in the absence of inhibitors
was used as the control (100%). System A uptake was calculated as[control uptake in Na +-PBS] – [uptake in the presence of MeAIB in
Na +-PBS]. Uptake by system ASC and/or other systems (for example
IMINO, B 0, G-like, N, and y +L which cannot be individually assessedyet) was calculated as [uptake in the presence of MeAIB in Na +-PBS]– [control uptake in Na + free-PBS]. System L uptake was calculated
as [control uptake in Na + free-PBS] – [uptake in the presence of
BCH in Na + free-PBS].

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S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

2.4. DNA microarray and quantitative real-time PCR
DNA microarray and quantitative real-time PCR were performed
using the method s of Okudaira et al. [25] and Yoshimoto et al. [26]The quality of RNA preparations was assessed using RIN (RNA Integrity
Number) software tool on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The expression levels of neutral aminoacid transporters in the four types of human carcinoma cells were analyzed using a DNA microarray (Agilent Technologies). The values are
expressed as relative quantities to a universal reference RNA (Stratagene Products Division, Agilent Technologies).
mRNA levels of neutral amino acid transporters were determined byconducting qRT-PCR assays. Ampliﬁcation and real-time ﬂuorescence
detection were performed using a model Mx3005P Real Time QPCR
system (Stratagene Products Division, Agilent Technologies). All data
were normalized using the geometric mean of β-actin and GAPDH. All
reactions were performed in triplicate.
3. Results
3.1. Uptake of [14C]MeAIB, [3H]MET and [3H]FLT transport in humancarcinoma cells
As shown in Fig. 1 for all four carcinoma cell lines the uptake of [3H]
MET under control conditions (Na +-PBS) was found to be about 3–5
times higher than the uptake of either [ 14C]MeAIB. The uptake of [ 14C]
MeAIB relative to [ 3H]FLT varied between the different cell lines with
[ 3H]FLT showing greater uptake than [ 14C]MeAIB in A431 and PC14
cells and [ 14C]MeAIB showing greater uptake than [ 3H]FLT in H441
and LS180 cells.
3.2. Competitive inhibition studies using [3H]MET and [14C]MeAIB
The majority of [ 3H]MET transport appeared to occur in a Na +independent manner in all four carcinoma cell lines (Fig. 2). The inhibitor BCH caused a large signiﬁcant inhibition of this Na+-independenttransport indicating the importance of system L. Although, the system
A inhibitor MeAIB showed signiﬁcant inhibition of Na +-dependenttransport in H441, PC14, and LS180 cells, the absolute magnitude of inhibition was very small relative to the magnitude of inhibition seen with
BCH. The contribution of system A to MET uptake is therefore small relative to the contribution of system L. By comparing the uptake in MeAIB
treated cells in the presence of Na + with uptake in control medium
lacking Na+ the relative contribution of system ASC and/or other systems to MET uptake can be calculated. The ASC system was found tohave a signiﬁcant contribution to MET transport in PC14 and LS180

Uptake (%U( uptaake/105 cells)

4.0

MET
MeAIB

3.5

3.3. Relative contribution of amino acid transport systems for MET and
MeAIB in human carcinomas
The relative contributions of amino acid transport systems A, L, and,
ASC/other to the uptake of [3H]MET and [ 14C]MeAIB in the four types ofhuman carcinomas were estimated using the modiﬁed methods of
Shikano et al. [23] and Nakajima et al. [24] and the results are shownin Table 1. Although the dominant uptake mechanism for [ 3H]MET inhuman carcinomas was through system L amino acid transport, system
A, ASC, or other Na +-dependent system(s) also played a role. The
dominant uptake mechanism of [ 14C]MeAIB in human carcinomas was
Na+-dependent and occurred via system A.
3.4. Correlation between [3H]MET and [14C]MeAIB uptake and transporter
gene expression or [ 3H]FLT in human-derived tumor cells
With regard to the contribution of system L amino acid transport
to the Na +-dependent uptake of [ 3H]MET the A431 and H441 cells
had the highest system L contribution (76.3%and 48.9% respectively)(Table 1). When the expression of numerous sodium-dependent and-independent amino acid transporters were analyzed using microarraygene expression proﬁling, as well as by qRT-PCR, the expression of thesystem L transporter LAT1 and the coupling factor 4F2hc were foundto be abundantly expressed in both A431 and H441 cells compared to
the PC14 and LS180 cells (Tables 2 and Table 3). Based on the resultsof the qRT-PCR analysis, the expression of LAT1 varied across the four
different carcinoma cell lines (Table 3). When the [ 3H]MET uptake inthe presence of Na + for all four carcinomas was plotted against total
LAT (system L) expression no correlation was found (Fig. 4A). On theother hand, there was a signiﬁcant correlation between accumulationof [ 14C]MeAIB and total SNAT (system A) gene expression in the carcinoma cells (Fig. 4B). Moreover, there are signiﬁcant correlations between accumulation of [ 3H]FLT and accumulation of [ 14C]MeAIB as
well as between [ 3H]FLT accumulation and gene expression level ofthe system A amino acid transporter SNAT in the carcinoma cells(Fig. 5C and D). For [ 3H]MET, the R 2 value between accumulation of[ 3H]FLT and accumulation of [ 3H]MET was lower compared with [ 14C]
MeAIB (Fig. 5A and C). There was no correlation between accumulationof [ 3H]FLT and gene expression of the total system L amino acid transporter (LAT) expression (Fig. 5B).

FLT

3.5. Statistical analysis

3.0
2.5

Data are presented as means and ± SDs. P values were calculatedusing a two-tailed paired Student t test for comparison between two
groups. A P value less than 0.05 was considered signiﬁcant.

2.0
1.5

4. Discussion

1.0
0.5
0

cells but not in A431 or H441 cells, although as for system A, the relative
magnitude was less than system L.
In contrast, in all four carcinomas, the majority of [14C]MeAIB uptake
was Na + dependent (Fig. 3) and as expected there was a large and signiﬁcant inhibition of this Na + dependent uptake of [ 14C]MeAIB by
MeAIB indicating the importance of system A. The system L inhibitor
had either no or minimal effect on the remaining Na +-independenttransport.

A431

H441

PC14

LS180

control / Na +-PBS
Fig. 1. Uptake of [14C]MeAIB, [3H]MET, and [3H]FLT by four human carcinoma cell lines.
Uptake of radiolabeled amino acid analogs in the presence of Na+ was measured 10 min
after addition to four different carcinoma cell lines. Data are expressed as %ID/105 cells.
Error bars represent S.D. (n = 4). ID injected dose.

The amino acid analog MeAIB is an inhibitor and speciﬁc substrate
for system A amino acid transport and the main transport mechanism
for MeAIB uptake is thought to occur via system A [27–32]. A study in
Chinese hamster ovary (CHO) cells showed that more than 90% of
MeAIB transport occurred through system A [9,33]. A study in cultured
human erythroleukemic (K562) cells using radiolabeled [ 14C]MeAIB
also gave the same result [34]. Given all of data, it is thought that system
A is the principal [ 11C]MeAIB transport pathway in in vivo human PET

S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

11

Control / Na+-PBS
MeAIB / Na+-PBS
NS
NS

4

Control / Na+ free-PBS**

BCH / Na+ free-PBS**

3.5

MET uptake (% uptaake/105 ccells)

**

**

3

**

**

NS

System A
2.5

*
**

*

System ASC and/or others
System L

2
1.5
1
0.5
0
A431

H441

PC14

LS180
*P<0.05**P<0.005

Fig. 2. Effect of Na+ and selective amino acid transport inhibitors on the uptake of [3H]MET by four human carcinoma cell lines. Uptake of [3H]MET by four different cell lines was measured
under the following conditions; (i) in the presence of Na+ (Na+-PBS) and either the absence or presence of the transport inhibitor MeAIB at a ﬁnal concentration of 1 mM (Control/Na+PBS and MeAIB/Na+-PBS respectively) and (ii) in the absence of Na+ (Na+-free PBS) and either the presence or absence of the transport inhibitor BCH at a ﬁnal concentration of 1 mM
(Control/Na+ free-PBS and BCH/Na+ free-PBS respectively). As shown for the PC14 cell line, system A uptake was calculated as [control uptake of [3H]MET in Na+-PBS] – [uptake of [3H]
MET in the presence of MeAIB in Na+-PBS]. Uptake by system ASC and/or other systems was calculated as [uptake of [3H]MET in the presence of MeAIB in Na+-PBS] – [control uptake of[3H]MET in Na+ free-PBS]. System L uptake was calculated as [control uptake of [3H]MET in Na+ free-PBS] – [uptake of [3H]MET in the presence of BCH in Na+ free-PBS]. Data are expressed
as %ID/105 cells. Error bars represent S.D. (n = 4). *P b 0.05 and **P b 0.005, NS not signiﬁcant. ID injected dose.

studies [19,20], although no human PET study has been conducted in
the presence of amino acid transport inhibitors. In our study using
four different types of human carcinoma cells we have shown thatsystem A contributes more than 70% to [ 14C]MeAIB uptake. In contrast
the uptake of [ 3H]MET was less speciﬁc; although system L was the predominant mechanism there were contributions of both system A andControl / Na+-PBS
MeAIB / Na+-PBS
Control / Na+ free-PBS

1.2

***
**

MeAIB
B uptakee (% upttake/105 cells)

***
***

1

*

***

0.8
0.6

BCH / Na+ free-PBS

System A
System ASC and/or others
System L

***

***

***

NS

0.4
***

0.2

NS

0
A431

H441

PC14
*P<0.05

LS180**P<0.01***P<0.005

Fig. 3. Effect of Na+ and selective amino acid transport inhibitors on the uptake of [14C]MeAIB by four human carcinoma cell lines. Uptake of [14C]MeAIB by four different cell lines wasmeasured under the following conditions; (i) in the presence of Na+ (Na+-PBS) and either the absence or presence of the transport inhibitor MeAIB at a ﬁnal concentration of 1 mM
(Control/Na +-PBS and MeAIB/Na +-PBS respectively) and (ii) in the absence of Na + (Na+-free PBS) and either the presence or absence of the transport inhibitor BCH at a ﬁnal
concentration of 1 mM (Control/Na+ free-PBS and BCH/Na+ free-PBS respectively). As shown for the A431 cell line, system A uptake was calculated as [control uptake of [14C]MeAIB
in Na+-PBS] – [uptake of [14C]MeAIB in the presence of MeAIB in Na+-PBS]. Uptake by system ASC and/or other systems was calculated as [uptake of [14C]MeAIB in the presence of
MeAIB in Na+-PBS] – [control uptake of [14C]MeAIB in Na+ free-PBS]. System L uptake was calculated as [control uptake of [14C]MeAIB in Na+ free-PBS] – [uptake of [14C]MeAIB in thepresence of BCH in Na+ free-PBS]. Data are expressed as %ID/105 cells. Error bars represent S.D. (n = 4). *P b 0.01, **P b 0.05, ***P b 0.005, NS not signiﬁcant. ID injected dose.

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S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

Table 1
Relative functional contributions of different amino acid transport systems in four humancarcinoma cells.

[3H]MET[14C]MeAIB

Cells

System A

System ASC and/or others

System L

A431
H441
PC14
LS180
A431
H441
PC14
LS180

–
15.8%
15.0%
13.7%
76.8%
76.1%
71.5%
73.9%

–
–
21.2%
13.7%
7.1%
9.8%
14.4%
10.5%

76.3%
48.9%
38.5%
38.5%
3.6%
–
–
–

Table 3Absolute quantiﬁcation of sodium-dependent and -independent amino acid.

Sodium dependent

SystemTransporter

A431

H441

PC14

LS180

A

SNAT1
SNAT2
SNAT4
ASCT1
ASCT2
y+LAT1y+LAT2
LAT1
LAT2
LAT3
LAT4
4F2hc

1.341
0.407
0.001
0.110
1.452
0
0.038
2.384
0.040
0
0.005
4.337

0.917
0.306
0
0.236
0.659
0
0.016
5.688
0.179
0.006
0.008
10.085

0.767
0.814
0
0.034
0.300
0.066
0.055
0.064
–
0.007
0.002
0.508

0.915
0.203
0.004
0.083
0.893
0.084
0.119
0.768
0.111
0.051
0.037
2.171

ASC
y +L
Sodium independent

L

System L cofactor

4F2hc

Percentage of control.

Copy/10 copies of housekeeping gene.

system ASC and the relative contributions of all systems varied between
the different cell lines (Table 1). Stability studies have shown that morethan 95% of [ 11C]MeAIB remained unchanged in human plasma 30 min
after administration. Since it is an unnatural amino acid, it also cannot
be used for protein synthesis [17]. Therefore, [ 11C]MeAIB imaging may
more accurately represent the uptake of amino acid transporter system
A compared to [ 11C]MET. Since [ 11C]MeAIB has higher selectivity forsystem A compared to [ 11C]-2-aminoisobutyric acid ([11C]AIB, anothersystem A substrate) [27,31], [ 11C]MeAIB is expected to be very usefulin system A amino acid transport PET imaging. We have previously reported the utility of [11C]MeAIB in the diagnosis of chest diseases, especially in the differential diagnosis between sarcoidosis and metastasis[21]. [ 11C]MeAIB has comparable sensitivity to [ 18F]FDG PET for the diagnosis of prostate cancer [35] and the tumor/non-tumor ratio obtainedwith [ 11C]MeAIB precisely distinguishes the malignant group from thebenign group in patients with brain tumors [36].

Table 2
Microarray gene expression proﬁling of sodium-dependent and -independent amino acidtransporters in four human carcinoma cell lines.

Sodiumdependent

SystemTransporter A431

H441

PC14

LS180

A

SNAT1

0.938
1.128
0.319
0.654
0.298
0.587
1.407
4.089
0.693
3.487
1.360
1.860
0.100
5.275
0.157
0.723
0.023
0.164
0.184
0.572
1.012
0.931
7.329
11.428
2.500
0.669
0.182
3.366
0.337
0.134
9.485
9.681

0.687
0.553
0.835
1.740
0.143
0.268
2.324
1.305
0.836
0.432
0.174
0.896
0.254
0.019
0.829
2.296
0.023
0.074
36.885
2.559
1.235
1.381
0.244
0.166
0.014
0.753
0.057
0.332
0.156
0.059
0.637
0.560

0.383
1.022
0.190
0.382
0.491
0.389
0.252
0.938
0.470
1.036
0.395
2.097
0.249
0.037
0.398
2.622
1.302
0.850
46.218
3.589
0.694
1.220
1.197
1.693
1.309
5.427
0.569
0.159
0.227
0.086
1.946
1.939

SNAT2
SNAT4

GLY
ASC

β

ASCT2
SNAT3
SNAT5
Taut

B0

B0AT

y +L

y+LAT1y+LAT2

L

LAT1

N

Sodiumindependent

Coupling factor

SNAT6
GlyT1
GlyT2
ASCT1

T
B0
asc
L, y+L, asc,
X-C

LAT2
LAT3
LAT4
TAT1
BAT1
asc1
4F2hc

1.233
1.148
0.448
1.022
0.148
0.286
3.860
5.004
0.575
1.355
0.644
3.536
0.285
10.941
0.348
1.102
0.047
0.080
0.167
1.882
0.618
0.942
2.700
4.981
0.540
0.193
0.106
0.116
0.166
0.065
4.682
4.646

On the other hand, the main transport mechanism of [ 3H]MET intumor cells has been reported to be system L [21,35], which is consistent
with our study results. The radiolabeled amino acid [ 11C]MET has the[ 11C] group incorporated into the methyl group attached to the sulfur.
[ 11C]MET is used at a lower rate in protein synthesis compared withother [ 11C] labeled amino acids such as [1- 11C]-L-methionine which
has the label in the carboxyl terminal. Therefore, [ 11C]MET tumor uptake represents a combination of amino acid transport and intracellular
metabolism [37]. However, since radiosynthesis of [ 11C]MET is extremely simple and easy, in clinical practice, [ 11C]MET-PET, together with [18F]
FET, has been widely used in brain tumor imaging [8,9].
We investigated the correlation between accumulation of [ 3H]MET
and [ 14C]MeAIB in four types of human carcinoma cells and gene expression, which led to the conclusion that there was no correlation between [ 3H]MET uptake and LAT expression (Fig. 4A). A weakrelationship between [3H]MET uptake and total LAT expression has previously been reported [22,26]. The reasons for this lack of correlation are
not completely clear but could perhaps be related to the fact that LAT1
biology is complex and involves a number of other proteins. One suchprotein is the amino acid transporter activating factor 4F2hc withwhich LAT1 forms disulﬁde-linked heterodimers thereby becoming active. A second protein is ASCT2. LAT1 transports neutral amino acid withlong side chains, such as leucine, and simultaneously counter transports
intracellular glutamine [38,39]. The intracellular glutamine supply ismaintained via the intracellular Na +-dependent amino acid transporter
ASCT2 that helps facilitate LAT1 function. The interplay between LAT1,
ASCT2, and 4F2hc is thought to be important in controlling cancer cell
metabolism [40]. For example, co-expression of LAT1 with ASCT2 hasbeen shown in lung cancer, and this co-expression, but not the sole expression of LAT1 or ASCT2, is strongly related to prognosis [41].
In our study, [ 14C]MeAIB accumulation in four types of human carcinoma cells was correlated with total SNAT expression (Fig. 4B). Since
SNAT does not require an activating factor, there is a high possibilityof a consistent relationship between SNAT expression and cellular uptake. System L, which transports amino acids into cells, has a countertransport mechanism associated with it. In contrast, system A, which
also transports amino acids into cells, has no counter transport activity.
Amino acid transporter function can therefore be evaluated directly
using MeAIB (system A substrate), allowing differentiation from system
L [42,43]. The correlation between the uptake of radiotracers and thegene expression of amino acid transporter in tumor cells has been evaluated in a number of recent nuclear medicine studies. A correlation between 4-borono-2-18F-ﬂuoro-phenylalanine ([18F]FBPA) and L-3-[18F]
ﬂuoro-α-methyl tyrosine ([ 18F]FAMT) uptake and LAT1 expression[15,16,26], a correlation between [S-methyl- 3H]-D-MET uptake and
LAT + ASCT expression [22], and a correlation between Trans-1amino-3-[ 18F]ﬂuorocyclobutanecarboxylic acid ([ 18F]FACBC) uptake
and ASCT expression [25,44] have all been recently reported. However,it should be borne in mind that gene expression levels may not directly
correlate with protein levels. In addition, cell-surface localization and

S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

A

13

B
MeAIB-SNAT

MET-LAT
1.0

MeAIB uptake(% uptake/105cells)

MET uptake(% uptake/105 cells)

4
3
R = 0.082

21

0.8
R = 0.883*

0.6
0.4
0.2
0

0
0

2

4

6

0

8

0.5

1.0

1.5

2.0

Gene expression of system A(copy / 10 copy of housekeeping gene)

Gene expression of system L(copy / 10 copy of housekeeping gene)*P<0.05
Fig. 4. Correlation between gene expression levels of system A and system L amino acid transporters and uptake of [3H]MET and [14C]MeAIB in human carcinomas. The ﬁgures show the
relationship between [3H]MET uptake and the gene expression levels of total amino acid transporter system L (total LAT) (A) and between [14C]MeAIB uptake and gene expression levels of
total amino acid transporter system A (total SNAT) (B). A431 (●), H441 (▲), PC14 (■) and LS180 (○). Tumor cell uptake of [14C]MeAIB showed a high correlation with the levels of gene
expression of amino acid transport system A. *P b 0.05.

B
FLT-MET
1.6
1.2
0.8

R = 0.765*

0.4
0

0

1

2

3

4

FLT uptake (% uptake/105 cells)

FLT uptake (% uptake/105 cells)

A

FLT-LAT
1.6
1.2
0.8

R = 0.361

0.4
0

0

5 cells)

MET uptake (% uptake/10

4

6

8

D
FLT-MeAIB
1.6
1.2
R = 0.915**

0.8
0.4
0

0

0.2

0.4

0.6

0.8

1.0

FLT uptake (% uptake/105 cells)

C
FLT uptake (% uptake/105 cells)

2Gene expression of system L(copy / 10 copy of housekeeping gene)

FLT-SNAT
1.6
1.2
0.8
0.4
0

MeAIB uptake (% uptake/105 cells)*P<0.05

R = 0.996***

0

0.5

1.0

1.5

2.0

Gene expression of system A(copy / 10 copy of housekeeping gene)**P<0.025***P<0.001

Fig. 5. Correlation of [3H]FLT uptake with either [3H]MET uptake, the gene expression levels of amino acid transporter system L, either [14C]MeAIB uptake or the gene expression levels ofamino acid transporter system A in human carcinomas. The ﬁgures show the relationship between [3H]FLT uptake and [3H]MET uptake (A), between [3H]FLT uptake and the gene
expression levels of total amino acid transporter system L (total LAT) (B), between [3H]FLT uptake and [14C]MeAIB uptake (C) and between [3H]FLT uptake and the gene expression
levels of total amino acid transporter system A (total SNAT) (D). A431 (●), H441 (▲), PC14 (■) and LS180 (○). Tumor cell uptake of [3H]FLT showed a high correlation with [14C]
MeAIB uptake and with the total gene expression levels of amino acid transport system A (total SNAT) and no correlation with the total gene expression levels of amino acid transportsystem L (total LAT). *P b 0.05, **P b 0.025, ***P b 0.001.

14

S. Kagawa et al. / Nuclear Medicine and Biology 49 (2017) 8–15

activity of amino acid transporter proteins may not necessarily be predicted by gene expression levels.
Our data also showed that [3H]FLT accumulation was strongly correlated with SNAT expression and [14C]MeAIB accumulation (Fig. 5C and
D). Uptake of 3′-deoxy-3′-[ 18F]ﬂuorothymidine ([18F]FLT), followed byphosphorylation by Thymidine Kinase 1 (TK1) leads to accumulationof the phosphorylated form of [ 18F]FLT in cells. The TK1 activity ofcells has been reported to be related to cellular proliferative activity,
which can be easily evaluated based on [ 18F]FLT accumulation[45–47]. Based on these data, since [ 3H]FLT accumulation is correlatedwith [ 14C]MeAIB accumulation, it is reasonable to conclude that [ 11C]
MeAIB accumulation is correlated not only with SNAT expression but
also with cellular proliferative activity. For [ 3H]MET, the R 2 value between accumulation of [ 3H]FLT and accumulation of [ 3H]MET waslower comparison with [ 14C]MeAIB (Fig. 5A and C). From the perspective of statistics, the correlation of [ 3H]FLT uptake needs to be examinedwith either [3H]MET uptake or [14C]MeAIB uptake in many human carcinoma cells. Studies using Ki-67 in many human carcinoma cells areunderway to conﬁrm the uptake results observed correlating [ 14C]
MeAIB and [ 3H]MET to [ 3H]FLT uptake.
Glucose metabolism, as well as amino acid metabolism, increases asa requirement to supply an energy source for cell proliferation in cancer
cells [48]. Moreover, amino acids and amino acid transporters play important roles other than in energy metabolism, such as in macromolecular synthesis, mTOR activation, and ROS homeostasis beyond energy
metabolism [49]. There are approximately ﬁfty different types ofamino acid transporters, but only LAT1 [50], LAT3 [51], ASCT2 [52],
ATB 0, +[53] and xCT [54] have been reported to be expressed at highlevels on the surface of cancer cells. Recently, there have been numerous
reports in cancer cells related to glutamine transport via ASCT2 andLAT1 [55–57]. Moreover, the expression levels of the SNAT amino acidtransporter, which belongs to the SLC38 family and like ASCT2 andLAT1 is related to glutamine transport, have been examined in a rangeof different cancers. Overexpression of SNAT was observed in gastriccancer [58], human hepatocellular carcinoma [59], breast cancer[60,61], hilar cholangiocarcinoma [62], C6 glioma [63], prostate cancer[25], HeLa epithelial cervical cancer cells, and 143B osteosarcoma cells[64]; and SNAT expression in stomach and breast cancers is also related
to Ki-67 [61] and PCNA (proliferating cell nuclear antigen) expression[58] as indicated by proliferative activity.
Reportedly, the accumulation of [ 11C]AIB, an unnatural amino acidand amino acid transporter system A substrate, accumulation in cancer
cells post-radiation therapy is correlated with changes in SLC38A1 expression [65]. Data from the Oncomine analysis (a gene expression database of 947 human cancer cell lines, http://www.broadinstitute.org/
ccle) revealed that ASCT2, SNAT1 and SNAT2 were overexpressed,
which could lead to a new treatment for preventing proliferation of cancer cells [64]. The studies reported here highlight the potential importance of SNAT in cancer. As described earlier, there have been an
increasing number of studies highlighting the role of SNAT in cancer
cells. Since [ 11C]MeAIB accumulation is correlated with SNAT expression and SNAT expression is correlated with [ 18F]FLT accumulation incancer cells, this suggests that there is a relationship with tumor proliferative activity.
Our results along with previous investigations support the concept that SNAT mediates the uptake of [ 11C]MeAIB by tumor cells.
We expect that accurate PCR studies examining the relationship between amino acid transporter gene expression and amino acid transporter activity in human carcinoma cells will be performed in the
future.

Acknowledgments
We wish to thank Hiroyo Araki and Kazuyo Ohe of Graduate
School of Medical Sciences, Kanazawa University, and Mariko Yuji of

Department of Radiology, Faculty of Medicine, University of Miyazaki
for their excellent technical assistance.

Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.nucmedbio.2017.01.008.

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